Diesel Engine Exhaust-Induced Alterations In DNA Methylation And Their Associations With Cytokinesis-Block Micronucleus Cytome Indexes

In recent years, concerns regarding the adverse health effects related to air pollutants exposure are increasing. Among numerous air pollutants, particle relates most closely with adverse health effects. Moreover, previous study has shown that fine particle may have more health impacts than coarse particle. Diesel engine exhaust (DEE) is one of the main sources of fine particle. Given the prevalence of diesel engine in urban district, a large number of urban residents are exposed to DEE in their daily lives. In addition, occupational population, such as miners and truck drivers, are also exposed to DEE during routine work. Epidemiological studies have shown that long-term exposure to DEE is associated with increased lung cancer risk. Genetic damage is recognized as a key event in the initiation of carcinogenesis.Cytokinesis-block micronucleus assay (CBMN) is frequently used to evaluate the extent of genetic damage in population exposed to genotoxic substance by measuring frequencies of micronucleus (MN), nucleoplasmic bridge (NPB), and nuclear bud (NBUD). Although it has been reported that DEE exposure can induce an increase in MN frequency, whether DEE exposure is associated with alterations in CBMN cytome indexes is not fully understood in humans due to presence of mixed exposure.In addition, the regulation patterns of biological pathways through which DEE exposure induces genotoxic effects or DNA damage response (DDR) is activated are still unclear. Epigenetics is heritable changes in gene expression that cannot be explained by changes in DNA sequence. More and more studies have demonstrated that epigenetic modifications as a novel regulation mechanism of gene expression play a pivotal role in the continuum of environmental exposure, toxic effects, and damage response. On the one hand, epigenetic modifications can reflect the extent of environmental exposure. On the other hand, epigenetic modifications can be involved in or respond to environmental exposure-induced genotoxic effects. Therefore, epigenetic modifications can serve as not only the biological mechanism mediating the association of environmental exposure with cancer risk, but also the biomarkers of environmental exposure and genetic damage. DNA methylation which refers to covalent addition of methyl group at 5-carbon of cytosine is the most widely studied epigenetic modification so far. In most cases, occurrence of DNA methylation ensues with gene silencing. The methylation status of DDR-related genes and repetitive element is crucial for maintaining genomic stability. Aberrant changes in methylation levels of DDR-related genes and repetitive element have been observed in lung cancer patients. Recently, more and more population studies regarding DEE exposure and altered DNA methylation have emerged. Acute exposure to DEE has been associated with altered methylation levels of genes involved in protein kinase and NFkB pathways in asthmatics. However, there still exists some critical scientific problems that are urgently needed to be solved, such as whether long-term exposure to DEE can cause methylation alterations in DDR-related genes and repetitive element and whether these changes in turn are associated with genotoxic effects induced by DEE exposure.To address the above problems, we recruited 117 diesel engine testing workers who are exclusively exposed to DEE and 112 non-DEE-exposed workers. We measured methylation levels of DDR-related genes and repetitive element as starting point and also detected CBMN cytome indexes. Our aim was to explore the relationships among DEE exposure, DNA methylation, and genotoxic effects.1. The analysis and characterization of DEE componentsScanning mobility particle size spectrometer and carbon analyzer were used to measure particle size distribution and carbon contents of DEE, respectively. Gas chromatography mass spectrometry (GC/MS) was used to measure polycyclic aromatic hydrocarbons (PAHs) contents in particle phase of DEE and personal samples of DEE-exposed workers. Portable GC/MS was used to measure concentrations of organics in gas phase of DEE. Passive samplers were used to measure concentrations of nitrogen dioxide and sulfur dioxide. High performance liquid chromatography-tandem mass spectrometry was used to measure urinary concentrations of 6 mono-hydroxylated PAHs (OH-PAHs) of workers. The results indicated that 84.3% of diesel exhaust particles had a size below 100nm. Elemental and organic carbon accounted for 28.6 ± 6.3% and 36.2 ± 6.5% of fine particle, respectively. The ratio of organic carbon to elemental carbon was 1.31 ± 0.31. Carcinogenic and non-carcinogenic PAHs accounted for 83.3% and 16.7% of total PAHs. The gas phase organics of DEE mainly consisted of alkanes and derivatives, cycloalkane derivatives, and benzene and derivatives. Elemental carbon showed a significant correlation with organic carbon (r=0.630,P=0.002) and borderline significant correlations with nitrogen dioxide and sulfur dioxide (r=0.370, P=0.090; r=0.385, P=0.077). The personal exposure levels to naphthalene, fluorene, phenanthrene, and pyrene and urinary concentrations of summed hydroxynaphthalene, 2-hydroxyfluorene, summed hydroxyphenanthrene, and 1-hydroxypyrene were all significantly higher in DEE-exposed workers than those in non-DEE-exposed workers.2. Associations of DEE exposure with DNA methylationPyrosequencing was used to measure the methylation levels of 3 DDR-related genes (i.e., p16, RASSF1A, and MGMT) and LINE-1 repetitive element in peripheral blood lymphocytes (PBLs) of workers. The results showed that DEE-exposed workers had significantly lower methylation levels of p16, RASSFIA, and MGMT than did non-DEE-exposed workers (P<0.001). The difference in LINE-1 methylation level was not significant between DEE and non-DEE exposed workers. Increasing quartiles of urinary summed OH-PAHs was associated with reduced methylation levels of p16, RASSFIA, and MGMT (Ptrend=0.018,0.006, and <0.001). In nonsmoking workers, after adjusting for potential confounding factors, an interquartile (IQR) increase in urinary summed OH-PAHs concentration was associated with 0.13,0.18, and 0.19 decrease in logit-transformed methylation level of p16, RASSFIA, and MGMT, respectively. In DEE-exposed workers, DEE exposure duration was significantly correlated with methylation levels of p16 and RASSF1A (r=0.293, P=0.001 and r=0.409, P<0.001). Compared with solvent control, the directions of metylation alterations in p16, RASSF1A, and MGMT of primary lymphocytes treated with organic extractions of DEE were generally consistent with those observed in DEE-exposed workers.3. Associations of DEE exposure with CBMN cytome indexesThe CBMN assay was used to measure frequencies of MN, NPB, and NBUD in PBLs of workers. The genomic instability index was calculated by integrating MN, NPB, and NBUD frequencies. The CBMN cytome index was calculated by integrating genomic instability index and previously reported apoptosis and necrosis frequencies. The results showed that DEE-exposed workers had significantly higher MN, NPB, NBUD frequencies, genomic instability index, and CBMN cytome index than did non-DEE-exposed workers (P<0.001). Increasing quartiles of urinary summed OH-PAHs was associated with increased MN, NPB, NBUD frequencies, genomic instability index, and CBMN cytome index (Ptrend<0.001). In nonsmoking workers, after adjusting for potential confounding factors, an IQR increase in urinary summed OH-PAHs concentration was associated with 93.48%,467.46%,160.39%, 128.64%, and 121.22% increase in MN, NPB, NBUD frequencies, genomic instability index, and CBMN cytome index. In DEE-exposed workers, after adjusting for potential confounding factors, an IQR increase in urinary summed OH-PAHs concentration was associated with 38.13% and 27.63% increase in MN frequency and CBMN cytome index.4. The relationships among DEE exposure, DNA methylation, DNA damage indexes, and CBMN cytome indexesFactor analysis, correlation analysis, mediation analysis, and interaction analysis were used to explore the relationships among indexes measured in the current study and previously reported indexes including olive tail moment (OTM) indicative of DNA strand damage and 1.N6-ethenodeoxyadenosine (εdA) and 3,N4-ethenodeoxycytidine (εdC) indicative of DNA oxidative damage. The results showed that p16. RASSF1 A, and MGMT methylation were negatively correlated with εdA (r=-0.187, P=0.008;r=-0.177,P=0.013; r=-0.251, P<0.001), OTM (r=-0.300, P<0.001; r=-0.305, P<0.001; r=-0.311, P<0.001), and genomic instability index (r=-0.183, P=0.007; r=-0.212, P=0.002;r=-0.244, P<0.001). p16 and RASSFIA methylation were positively correlated with nuclear division index (r=0.220, P=0.001; r=0.225, P=0.001). Hypomethylation of p16 were found to mediate 24.9% and 6.1% of the associations of nuclear division index with OTM and genomic instability index, respectively. Hypomethylation of RASSFIA were found to mediate 26.1% and 6.6% of the associations of nuclear division index with OTM and genomic instability index, respectively. In nonsmoking workers, a one-unit increase in In-transformed urinary summed OH-PAHs was associated with 64.38% and 45.50% increase in genomic instability index for workers with LINE-1 methylation level <85.6% and >85.6%, respectively (Pinteraction=0.016). Genomic instability index was positively correlated with εdA and OTM (r=0.204, P=0.004;r=0.353,P<0.001). Using the lowest and highest quartiles of urinary summed OH-PAHs and εdA concentrations as the cutoff values for discrimination of DEE exposure and genetic damage levels, the area under curve of MGMT methylation were 0.758 and 0.711, respectively. Using the lowest and highest quartiles of OTM as the cutoff values for discrimination of genetic damage level, the area under curve of p16 and RASSFIA were 0.705 and 0.724, respectively.Conclusions1) The size of most diesel exhaust particles fell within the range of ultrafine particle. The PAHs adsorbed to diesel exhaust particles mainly comprised of carcinogenic PAHs. The gas phase organics of DEE mainly consisted of alkanes, benzene, and their derivatives. Elemental carbon showed significant or borderline significant correlations with other main components of DEE. The DEE-exposed workers had significantly higher urinary OH-PAHs concentrations than did non-DEE-exposed workers. With the increase of personal exposure levels to PAHs, the corresponding urinary OH-PAHs also increased in DEE-exposed workers.2) DEE exposure could induce decreases in methylation levels of p16, RASSFIA, and MGMT. DEE exposure was associated with hypomethylation of p16, RASSFIA, and MGMT. DEE exposure duration was positively correlated with p16 and RASSF1A methylation. MGMT methylation might serve as biomarker of DEE exposure.3) DEE exposure could induce increases in MN, NPB, NBUD frequencies, genomic instability index, and CBMN cytome index in PBLs. DEE exposure was associated with increased MN, NPB, NBUD frequencies, genomic instability index, and CBMN cytome index. DEE exposure level was associated with increased MN frequency and CBMN cytome index.4) p16, RASSF1A, and MGMT methylation were negatively correlated with εdA, OTM, and genomic instability index. Genomic instability index was positively correlated with εdA and OTM. p16 and RASSF1A methylation might serve as biomarker of DEE exposure-related OTM. MGMT methylation might serve as biomarker of DEE exposure-related εdA.5) p16 and RASSF1A methylation could mediate the associations of nuclear division index with OTM and genomic instability index. LINE-1 methylation could modify the association of DEE exposure with genomic instability index.